Method and apparatus for determining gas concentration

ABSTRACT

A method and an apparatus for measuring the concentration of a specific gas component of a gas mixture including another gas whose concentration is independently known using light absorption spectroscopy are provided. A method and an apparatus for assessing human airway inflammation by measuring the concentration of exhaled NO and CO 2  present in orally exhaled breath using light absorption spectroscopy are also provided. NO concentration is determined at the time during breath sampling corresponding to a known exhaled CO 2  concentration. Methods and apparatus are further provided for measuring NO concentration in orally exhaled human breath that analyze breath emanating from the lower airways and lungs, while excluding breath from the nasal cavity. They include steps and apparatus for discarding initially exhaled breath, flowing breath through an analysis chamber using a vacuum pump and flowing breath through an analysis chamber using a vacuum pump at an initial flow rate and later at a flow rate higher than the initial flow rate.

[0001] This application claims the benefit of U.S. Patent Application No. 60/347,513 filed Jan. 11, 2002.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates to methods and apparatus for determining the concentration of a gas in a mixture.

[0004] 2. Description of the Prior Art

[0005] Gas mixtures are routinely analyzed in fields ranging from medical diagnostics to automobile design. Techniques used vary widely and include mass spectroscopy, infrared spectroscopy, chemiluminescence and flame ionization. Regardless of the application, accuracy, reliability, convenient operation, low cost and preferably real-time analysis results are desirable. In the field of medical diagnostics, convenient, patient-friendly operation and real-time delivery of results are crucial for successful clinical implementation.

[0006] Currently available methods for determining the concentration of a gas in a mixture typically require use of a reference as in U.S. Pat. No. 5,640,014 to Sauke et al. wherein a reference signal or reference gas is used in a diode laser spectroscopy method to determine the isotopic ratio of a gas in a sample. Other methods involve deliberate introduction of a trace gas to the gas mixture to be analyzed as in U.S. Pat. No. 6,412,333 to Inoue et al. U.S. Pat. No. 6,412,333 describes an auto exhaust analyzer system and method wherein a trace gas is introduced into the exhaust gas stream, the mass of the trace gas is calculated using the analyzer and the calculated mass then compared with the known mass of trace gas to verify the accuracy of the analysis. Still other techniques compensate for inaccuracy in non-dispersive infrared analysis using signal processing techniques such as U.S. Pat. No. 5,464,983 to Wang which describes measurement of change of signal (CS) and change of change of signal (CCS) and comparison of CCS data obtained to CCS data for known gases at known concentrations and temperatures.

[0007] In the field of medical diagnostics, nitrogen monoxide (NO) is a well-established indicator of pulmonary function. Analysis of exhaled nitric oxide provides a health care provider with a non-invasive test for inflammatory diseases of the lower airways such as asthma. Widely used diagnostic tests for asthma such as spirometry give only limited and indirect information about lower airway inflammation. Current methods measuring exhaled NO for assessing lung function, such as U.S. Pat. No. 5,447,165 to Gustafsson, use mass spectroscopy and chemiluminescence to measure the time distribution of NO formed during exhalation and require that results be compared with unimpaired lung function of a living subject reference. U.S. Pat. No. 6,099,480 to Gustafsson describes collection of human breath and analysis for NO content using chemiluminescence or reagent-based chemical analysis techniques. U.S. Pat. No. 6,419,634 to Gaston et al. uses reagent-based calorimetric NO assay analysis of exhaled human breath condensate to evaluate airway inflammation.

[0008] Endogenous nitric oxide emanating from the airways and lungs is the preferred indicator of airway inflammation. Hence, breath collection techniques to collect this endogenous nitric oxide while excluding exhaled nasal NO from study have been developed. U.S. Pat. No. 6,010,459 to Silkoff et al. requires a patient to exhale at a constant rate and increases pressure in the mouth to close off the nasopharynx during exhalation thereby excluding nasal NO. U.S. Pat. No. 6,038,913 to Gustafsson et al. requires that a patient exhale against a back pressure during the later phase of an exhalation. U.S. Pat. No. 5,922,610 to Alving et al. describes a face mask that tightly covers the nose and/or mouth of the subject. Such techniques that require controlled exhalation by a patient can be difficult for those patients with impaired respiratory function and especially for pediatric patients. All of these methods require frequent calibration of the NO sensor using a calibration gas with a known NO concentration. This requirement complicates use of such sensing technology in a clinical setting.

[0009] Thus, there exists a need for accurately determining the concentration of a specific gas of interest in an as-obtained gas mixture sample without introduction of any additional reference gas. There exists further need for accurately assessing airway inflammation, using a single exhaled breath from a single patient without any healthy lung function reference. Preferably, such airway inflammation assessment is conveniently conducted in a clinical setting, maximizes patient comfort and provides real-time results that can enable a health care provider to diagnose and treat a patient in a single clinic visit

SUMMARY OF THE INVENTION

[0010] The invention provides a method for measuring the concentration of a specific gas of interest, such as a marker gas, in an as-obtained gas mixture sample that initially includes the gas of interest and an inherently present calibration gas whose concentration is independently known. The gas mixture sample is obtained and then placed in the gas sample cell of a spectrometer including a spectrometer light source, a spectrometer gas sample cell, a spectrometer detector and a spectrometer computer. The gas mixture sample is illuminated with the spectrometer light source and a transmitted light beam which has passed through the gas mixture sample is detected with the spectrometer detector to produce a detector electronic signal sequence. The detector electronic signal sequence is transmitted to the spectrometer computer which is programmed to acquire the detector electronic signal sequence for a signal acquisition interval to obtain a gas mixture spectrum. The gas mixture spectrum is stored and analyzed using the spectrometer computer. The analysis further includes identifying a marker gas absorption feature and a calibration gas absorption feature, determining a marker gas absorption feature intensity and a calibration gas absorption feature intensity, storing the marker gas absorption feature intensity and storing the calibration gas absorption feature intensity as a function of time over a gas mixture sampling interval. The steps of acquiring and analyzing are repeated for multiple signal acquisition intervals to identify a single signal acquisition interval wherein a known concentration calibration gas absorption feature which corresponds to an independently known calibration gas concentration and a simultaneous marker gas absorption feature occur. Then a ratio of simultaneous marker gas absorption feature intensity to known concentration calibration gas absorption feature intensity is calculated. This ratio is multiplied by a proportionality constant to determine marker gas concentration.

[0011] A method for assessing human airway inflammation by spectroscopically measuring NO, a marker gas associated with airway inflammation, in orally exhaled human breath is also provided. CO₂, a gas inherently present with the NO in the orally exhaled human breath and whose concentration is independently known, is simultaneously measured so that its known concentration can be used to calculate NO concentration. According to the method, an orally exhaled human breath sample including NO and CO₂ is collected and placed in a spectrometer gas cell which is part of a spectrometer that also includes a spectrometer light source, spectrometer detector and spectrometer computer. The orally exhaled human breath sample is illuminated with the spectrometer light source and a transmitted light beam that has passed through the orally exhaled human breath sample is detected with the spectrometer detector to produce a detector electronic signal sequence. The spectrometer computer acquires the detector electronic signal sequence for a signal acquisition interval to obtain a human breath sample spectrum. The spectrometer computer analyzes the human breath sample spectrum by identifying an exhaled NO absorption feature and an exhaled CO₂ absorption feature, determining an exhaled NO absorption feature intensity and an exhaled CO₂ absorption feature intensity, and storing exhaled NO absorption feature intensity and exhaled CO₂ absorption feature intensity as a function of time. The acquiring and analyzing steps are repeated for multiple signal acquisition intervals to identify a single signal acquisition interval wherein a known concentration CO₂ gas absorption feature which corresponds to an independently known CO₂ gas concentration and a simultaneous NO absorption feature occur. A ratio of simultaneous NO absorption feature intensity to the CO₂ gas absorption feature intensity corresponding to a known CO₂ concentration is calculated. This ratio is multiplied by a proportionality constant to obtain concentration of exhaled NO, thereby providing an assessment of human airway inflammation.

[0012] Another aspect of the invention provides an apparatus for measuring the concentration of a specific gas of interest, such as a marker gas, in an as-obtained gas mixture sample that initially includes the gas of interest and an inherently present calibration gas whose concentration is independently known. The apparatus includes a spectrometer gas sample cell to contain the gas mixture sample and a spectrometer light source to illuminate the gas mixture sample. A spectrometer detector detects light transmitted through the gas mixture sample so that a detector signal sequence is generated. The apparatus further includes a spectrometer computer embodying an executable program for repeatedly acquiring and analyzing the detector electronic signal sequence for multiple signal acquisition intervals to obtain gas mixture spectra which are then stored. The computer is further programmed to analyze the gas mixture spectra by identifying a marker gas absorption feature and a calibration gas absorption feature, and determine a marker gas absorption feature intensity and a calibration gas feature intensity. The program then calculates marker gas absorption feature intensity and calibration gas absorption feature intensity as a function of time to identify a single signal acquisition interval wherein a known concentration calibration gas absorption feature which corresponds to an independently known calibration gas concentration and a simultaneous marker gas absorption feature occur. A ratio of simultaneous marker gas absorption feature intensity to known concentration calibration gas absorption feature intensity is calculated and multiplied by a proportionality constant to determine concentration of the marker gas.

[0013] Additionally, an apparatus for assessing human airway inflammation by measuring NO in exhaled human breath is provided. The apparatus includes an executable computer program for analyzing NO light absorption. The computer program also analyzes the light absorption of CO₂, a gas inherently present with the NO in the orally exhaled human breath. Since the concentration of CO₂ is independently known, the computer program uses its known concentration to calculate NO concentration. The apparatus further includes a spectrometer gas sample cell containing an orally exhaled human breath sample. The human breath sample further includes NO and CO₂. A spectrometer light source for illuminating the orally exhaled human breath sample and a spectrometer detector for detecting light transmitted through the orally exhaled human breath sample and for generating a detector signal sequence is also provided. A spectrometer computer includes an executable program for analyzing NO and CO₂ light absorption by repeatedly acquiring and analyzing the detector electronic signal sequence for multiple signal acquisition intervals to obtain human breath sample spectra. The computer program stores the human breath sample spectra and analyzes them by identifying an exhaled NO absorption feature and an exhaled CO₂ absorption feature, determining an exhaled NO absorption feature intensity and an exhaled CO₂ absorption feature intensity. The program stores the exhaled NO absorption feature intensity and the exhaled CO₂ absorption feature intensity as a function of time. The computer program acquires and analyzes human breath sample spectra until a single signal acquisition interval is identified whose CO₂ absorption feature intensity corresponds to an independently known CO₂ concentration. The computer program calculates a ratio of the CO₂ absorption feature intensity corresponding to the known CO₂ concentration to NO absorption feature intensity obtained during that same single signal acquisition interval. This ratio is multiplied by a proportionality constant to obtain the concentration of NO, which provides an assessment of human airway inflammation.

[0014] A method for measuring NO concentration in orally exhaled human breath includes steps of having a human subject normally exhale a single breath into a mouthpiece. The first part of the single breath, initially exhaled human breath, is collected in a discard container and is discarded. The remainder of the single breath, the remaining orally exhaled human breath, is flowed to an NO analysis system where NO concentration is obtained through a tube connected to a vacuum pump.

[0015] An apparatus for measuring NO concentration in orally exhaled human breath including a mouthpiece for accepting orally exhaled human breath made up of initially exhaled human breath and remaining exhaled human breath is provided. The mouthpiece is connected to a discard container and to a flow tube, which is further connected to a vacuum pump and to a light absorption spectrometer system so that initially orally exhaled human breath is collected in the discard container and the remaining orally exhaled human breath is delivered to an NO analysis system where NO concentration is measured.

[0016] According to another method for measuring NO concentration in orally exhaled human breath, a human subject normally exhales a single breath, including initial orally exhaled human breath and remaining orally exhaled human breath into a mouthpiece. The single human breath is flowed through a tube connected to a vacuum pump and to an NO analysis system where the single human breath is analyzed to obtain NO concentration.

[0017] Another apparatus for measuring NO concentration in orally exhaled human breath includes a mouthpiece for accepting orally exhaled human breath, having initially exhaled human breath and remaining orally exhaled human breath from a human subject. The mouthpiece is connected to a vacuum pump and to an NO analysis system for obtaining NO concentration.

[0018] According to yet another method for measuring NO concentration in orally exhaled human breath, a human subject normally exhales a single breath into mouthpiece. The single breath is flowed from the mouthpiece to an NO analysis system through a tube connected to a vacuum pump and to the NO analysis system first at a first flow rate and later at a second flow rate, less than the first flow rate. The single breath is analyzed with the NO analysis system to obtain NO concentration.

[0019] Yet another apparatus is provided for measuring NO concentration in orally exhaled human breath. A mouthpiece for accepting a single orally exhaled human breath is connected to a flow tube. The flow tube is further connected to a vacuum pump and to an NO analysis system for obtaining NO concentration. The flow tube includes a flow rate controller for sequentially generating a first flow rate and a second flow rate less than the first flow rate.

[0020] Objects of the invention are to provide a method and apparatus for measuring the concentration of a gas of interest, a marker gas, in a gas mixture that inherently includes the marker gas as well as another gas, a calibration gas having an independently known concentration by obtaining and comparing spectroscopic absorption feature intensities for the marker gas and calibration gas corresponding to when the concentration of the calibration gas is known independently.

[0021] Other objects of the invention are to provide a method and apparatus for assessing human airway inflammation by measuring NO concentration in orally exhaled human breath. The CO₂ inherently present with NO in human breath has an independently known concentration and by obtaining and comparing spectroscopic absorption feature intensities for NO and CO₂ when the concentration of the CO₂ is known, NO concentration can be obtained.

[0022] Yet other objects of the invention are to provide a method and apparatus to measure NO concentration in orally exhaled human breath wherein the initial portion of a single orally exhaled breath is discarded so that the patient need only provide a single breath exhaled normally through the mouth.

[0023] Additional objects of the invention are to provide a method and apparatus to measure NO concentration in orally exhaled human breath wherein a single breath is delivered to a light absorption spectrometer system through a tube at a reduced pressure provided by a vacuum pump so that the patient need only provide a single breath exhaled normally through the mouth.

[0024] Further objects of the invention are to provide a method and apparatus to measure NO concentration in orally exhaled human breath wherein a single breath is delivered to an NO analysis system through a tube at a reduced pressure provided by a vacuum pump at two different flow rates, a first flow rate and then at a second flow rate less than the first flow rate so that the patient need only provide a single breath exhaled normally through the mouth.

[0025] Other and further objects, features, and advantages of the present invention will be readily apparent to those skilled in the art upon reading the description of the preferred embodiments which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 is a block diagram summarizing steps of acquiring and analyzing gas mixture spectra to determine concentration of a gas mixture component.

[0027]FIG. 2 is a schematic drawing of a gas mixture analysis system.

[0028]FIG. 3 is a plot of nitric oxide (NO) and carbon dioxide (CO₂) absorption line intensities in the 5.2 μm region of the infrared spectrum using data from the HITRAN '96 database.

[0029]FIG. 4 is a schematic drawing of a breath collection and analysis apparatus and the TLAS system equipped with a IV-VI mid-infrared laser.

[0030]FIG. 5 is a schematic drawing of a breath collection and analysis apparatus including a container for collecting initially exhaled breath.

[0031]FIG. 6 is a schematic drawing of a breath collection and analysis apparatus.

[0032]FIG. 7 is a schematic drawing of a breath collection and analysis apparatus including a flow controller.

[0033]FIG. 8 is a second harmonic spectrum of exhaled alveolar-enriched human breath measured between 1912.5 cm⁻¹ and 1913.0 cm⁻¹ showing absorption features for NO and CO₂.

[0034]FIG. 9 is gas calibration curve obtained by measuring concentrations of NO produced using a gas dilution system.

[0035]FIG. 10 is a plot showing measured second harmonic absorption features of NO (1912.79 cm⁻¹) for NO concentrations of 50 ppb, 124 ppb, 244 ppb, and 475 ppb.

[0036]FIG. 11 is a plot showing two comparison spectra (Comp. #1 and Comp. #2) at varying unknown concentrations of NO and CO₂.

[0037]FIG. 12 is a plot showing exhalation trends over a 20 second exhalation of eNO and eCO₂ measured from an asthmatic subject and a nonasthmatic subject.

[0038]FIG. 13 is a plot showing absorption magnitude of NO vs. time for 100 ppb NO gas flowed through a spectrometer gas sample cell for 20 seconds and for a subject's breath for a 20 second exhalation.

[0039]FIG. 14 is a plot of calibrated eNO breath measurements quantified using a 50 ppb NO gas standard versus eNO concentrations calculated using the eCO₂ absorption magnitudes and Equation (3).

[0040]FIG. 15 is a plot showing absorption magnitudes of eNO and eCO₂ as a function of time for varying exhalation times of 5, 10 and 15 seconds.

[0041]FIG. 16 is a plot of exhalation trends for eCO₂ at varying gas sample cell pressures between 13 Torr (1.7 kPa) and 33 Torr (4.4 kPa).

[0042]FIG. 17 is a plot of calculated eNO concentrations obtained using Equation (3) at varying pressures between 13 Torr and 40 Torr along with the eNO concentration mean, μ_(eNO), and standard deviation, σ_(eNO).

[0043] FIGS. 18(a) and 18(b) are plots showing breath eNO and eCO₂ exhalation trends measured from a nonasthmatic subject at different ambient NO levels.

[0044]FIG. 19 is a plot showing concentrations of eNO obtained using Equation (3) over a period of 10 days for an asthmatic patient undergoing a corticosteroid treatment regimen and for a non-asthmatic subject over the same time period.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0045] The invention provides a method and apparatus for spectroscopically measuring the concentration of a particular marker gas in a gas mixture using another gas inherently present in the mixture and whose concentration is known independently as an internal calibration gas. Thus, the invention obviates the need for a separate reference or for deliberate introduction of any reference or trace gas to the gas mixture.

[0046]FIG. 2 shows a preferred embodiment of the invention. A gas mixture sample, represented by arrow 10 is introduced via tubing 12 into a spectrometer sample cell, specifically White cell 14. A Herriott cell can also be used as the spectrometer sample cell. Mechanical vacuum pump 16 evacuates White cell 14 to keep White cell 14 at a pressure selected to reduce line broadening and interference between the spectroscopic absorption features associated respectively with the marker gas and calibration gas. Gas mixture sample 10 can be human breath which further includes a biomarker gas such as NO, H₂O₂, CO, CO₂, CS₂, C₂H₆, CH₃O, NH₃, H₂S, or OCS whose presence and concentration can be correlated with a disease. Calibration gases present in a human breath sample can be CO₂, H₂O, N₂O or CH₄. The spectrometer can be a mid-infrared tunable laser absorption spectroscopy system where the spectrometer light source that illuminates the gas mixture sample is IV-VI diode laser 18 with an emission wavelength in the range of from about 3 μm to about 10 μm controlled by current driver/function generator assembly 19 and personal computer 30 as shown schematically by arrow 17.

[0047] Transmitted light beam 20 that has passed through gas mixture sample 10 in White cell 14 can be focused with lens 22 onto spectrometer detector 24 which is maintained with IV-VI diode laser 18 at reduced temperature in cryostat 26. Cryostat 26 is evacuated using ion pump 28. Spectrometer detector 24 generates a signal sequence which can be acquired by lock-in amplifier 29 after amplification by a preamp (not shown) and personal computer 30, equipped with an analog-to-digital (A/D) card to produce a gas mixture spectrum. The gas mixture spectrum can be a second harmonic absorption spectrum. Arrow 15 represents signals transmitted from lock-in amplifier 29 to personal computer 64. The gas mixture spectrum can be generated by digitally filtering a running co-average of at least two detector signal sequences which have been obtained and stored in personal computer 30 as stored detector electronic signal sequences.

[0048] The invention further provides a method and apparatus for assessing human airway inflammation by measuring the concentration of NO, a biomarker gas associated with human body tissue inflammation. The NO is present together with CO₂ in orally exhaled human breath. Since the concentration of CO₂ in orally exhaled human breath is known, it serves as an internal calibration gas. NO and CO₂ spectroscopic absorption feature intensities are identified, obtained and stored repeatedly to find a signal acquisition interval wherein the CO₂ gas absorption feature intensity corresponds to a known CO₂ concentration. The CO₂ gas absorption feature intensity that corresponds to the independently known CO₂ concentration is the maximum CO₂ gas absorption feature intensity obtained over the entire time a human subject exhales, the exhalation interval. The NO gas absorption feature intensity obtained simultaneously with the CO₂ gas absorption feature intensity corresponding to an independently known CO₂ concentration in the range of from about 4% to about 5% is subsequently used in a ratio calculation to obtain the NO concentration which is an indicator for human airway inflammation. Exhaled NO concentration in the range of from about 5 parts per billion (ppb) to about 100 parts per billion (ppb)is indicative of airway inflammation.

[0049] As shown in FIG. 4, human subject 40 orally exhales so that orally exhaled breath enters spectrometer system 41. Human subject 40 orally exhales into mouthpiece 68. Mouthpiece 68 is connected with T-piece 66, while T-piece 66 is further connected to discard container 70 and tubing 72. A one-way flutter valve, not shown, can be connected between T-piece 66 and discard container 70 to keep initially orally exhaled breath in discard container 70. All connections allow for passage of orally exhaled human breath. Tubing 72 carries orally exhaled breath to gas sample cell 54. Spectrometer gas sample cell 54, which can be a Herriott cell or multi-pass White cell, can be kept at a pressure chosen to reduce line broadening as well as interference between absorption features associated, respectively, with the NO and CO₂ present in the orally exhaled breath sample. Human subject 40 can orally exhale a single breath as he or she would normally do while breathing not under test conditions so that breath provided at the beginning of the exhalation, initial exhaled breath, is collected in discard container 70 and is not analyzed by the spectrometer. After discard container 70 is filled, the rest of the breath from the single breath, remaining exhaled breath, enters spectrometer gas sample cell 54 for spectroscopic analysis. Orally exhaled human breath can also be introduced into spectrometer gas sample cell 54 using mechanical vacuum pump 65 connected to spectrometer gas sample cell 54 by tubing 63 alone or together with the already described discard container 70 to induce flow of the orally exhaled human breath through spectrometer gas sample cell 54. The flow rate of orally exhaled human breath through spectrometer gas sample cell 54 can be varied using flow rate controllers not shown so that the breath first flows through spectrometer gas sample cell 54 at a first flow rate greater than a later, second flow rate.

[0050] Spectrometer system 41 can be a mid-infrared tunable laser absorption spectroscopy system and can have a IV-VI diode laser 42 with an emission wavelength in the range of from about 3 μm to about 10 μm as the light source to illuminate the orally exhaled human breath sample contained in gas sample cell 54. Spectrometer detector 56 generates detector electronic signal sequences in response to detection of light beam 53 that has been transmitted through the orally exhaled human breath sample contained in gas sample cell 54. Detector electronic signal sequences are acquired by lock-in amplifier 60 after amplification by a preamp (not shown) spectrometer computer 64 to generate a human breath sample spectrum which can be a second harmonic absorption spectrum. Arrow 62 represents signals transmitted from lock-in amplifier 60 to personal computer 64. A running co-average of at least two previously obtained and stored detector electronic signal sequences can be performed and the result digitally filtered to generate the human breath sample spectrum.

[0051] The invention also provides a method and apparatus for measuring NO concentration in orally exhaled human breath generally applicable for use with a wide variety of analysis systems capable of measuring NO concentration utilizing a discard container to eliminate initial exhaled breath from analysis. FIG. 5 shows human subject 80 orally exhaling a single breath as he or she would do when breathing normally, into mouthpiece 82 connected via T-piece 84 to discard container 86 and flow tube 88. Breath provided at the beginning of the exhalation, initial exhaled breath, is collected in discard container 86 and is discarded, i.e., not analyzed. After discard container 86 is filled, the rest of the breath from the single breath, remaining exhaled breath, enters NO analysis system 90 through flow tube 88. Mechanical vacuum pump 92 is connected to NO analysis system 90 and flow tube 88 so that orally exhaled human breath flows through the NO analysis system. Mechanical vacuum pump 92 can be operated to produce a vacuum in the range of from about 10 Torr to about 80 Torr. The flow rate resulting from pumping on the system with mechanical vacuum pump 92 can be in the range of from about 0.5 liters per minute to about 30 liters per minute. NO analysis system 90 can be a light absorption spectrometer, such as a mid-infrared tunable laser absorption spectrometer.

[0052] The invention provides a method and apparatus to measure NO concentration in orally exhaled human breath wherein a single breath is delivered to an NO light absorption spectrometer system through a tube maintained at a reduced pressure provided by a vacuum pump so that the patient need only provide a single breath exhaled normally through the mouth. FIG. 6 shows human subject 100 orally exhaling a single breath as he or she would do when breathing normally into mouthpiece 102 connected to flow tube 104. All orally exhaled breath enters NO light absorption spectrometer analysis system 106 through flow tube 104. Mechanical vacuum pump 108 is connected to NO light absorption spectrometer system 106 and flow tube 104 so that orally exhaled human breath flows through the NO light absorption spectrometer system. Mechanical vacuum pump 108 can be operated to produce a vacuum in the range of from about 20 Torr to about 80 Torr. The flow rate resulting from pumping on the system with mechanical vacuum pump 108 can be in the range of from about 0.5 liters per minute to about 30 liters per minute. NO light absorption spectrometer system 106 can be a mid-infrared tunable laser absorption spectrometer.

[0053] The invention provides a method and apparatus to measure NO concentration in orally exhaled human breath wherein a single breath is delivered to an NO analysis system through a tube at a reduced pressure provided by a vacuum pump at two different flow rates, a first flow rate and then at a second flow rate less than the first flow rate so that the patient need only provide a single breath exhaled normally through the mouth. The flow rates can be adjusted using the spectrometer computer. The time that the first flow rate is used can be in the range of from about 0.1 sec to about 10 sec. The second flow rate can be used for a time in the range of from about 5 sec to about 20 sec.

[0054]FIG. 7 shows human subject 120 orally exhaling a single breath as he or she would do when breathing normally into mouthpiece 122 connected to flow tube 124. Mechanical vacuum pump 126 is connected to NO analysis system 128 and flow tube 124 so that orally exhaled human breath flows through NO analysis system 128. Flow controller 130 can be adjusted using a computer in NO analysis system 128 to provide a desired flow rate. The first flow rate is in the range of from about 2 liters per minute to about 30 liters per minute and the second flow rate is in the range of from about 0.5 liters per minute to about 20 liters per minute. NO analysis system 128 can be a light absorption spectrometer, such as a mid-infrared tunable laser absorption spectrometer.

EXAMPLE 1

[0055] The following example describes a liquid-N₂-free TLAS system equipped with a IV-VI laser operating near 5.2 μm for the purpose of analyzing eNO and exhaled CO₂ (eCO₂) simultaneously in expired breath. The system required no consumables other than disposable mouthpieces for breath analysis. Absorption measurements were performed using a 107-meter multipass White cell with a 16-liter volume. A closed-cycle cryogenic refrigerator was used to maintain cryogenic laser operating temperatures below 120 K. These refrigerators can dissipate about 5 watts of power at typical laser heat sink temperatures of ˜90 K. IV-VT lasers are well suited for cooling with such a system since they typically generate less than 1 watt of waste heat. The system further takes advantage of the ability of a single IV-VI laser to measure H₂O, CO₂, and NO simultaneously, thereby eliminating any need for introduction of additional calibration gases not originally present in the sample, reference cells, and reference detectors. A breath collection apparatus was fabricated to collect and sample breath in close accordance with the recommendations of the American Thoracic Society. Daily breath measurements from 5 individuals over a period of ten working days were performed. Daily eNO concentrations measured from the 5 individuals calculated using eCO₂ end-tidal absorption magnitudes as a reference are compared to concentrations obtained by comparison with a calibrated NO gas standard. The effect of elevated NO levels in the ambient air on calculated eNO concentrations using eCO₂ as an internal reference was also studied. To test the flexibility of the internal calibration scheme and to simulate measurements of a child's breath, an adult's breath was measured at different exhalation times from 5 to 20 seconds.

[0056] A brief discussion of specific absorption line attributes for the molecules of interest (NO, CO₂, and H₂O) between 1912.5 cm⁻¹ and 1913.0 cm⁻¹ is given. The R(10.5) NO lines located between 1912.7937 cm⁻¹ and 1912.7956 cm⁻¹ have a maximum absorption intensity of 1.032×10⁻²⁰ cm⁻¹/(molecule·cm⁻²) and are separated by 0.05 cm⁻¹ from the nearest H₂O and CO₂ absorption lines. The single CO₂ absorption line P(6) at 1912.96 cm⁻¹) was measured and has a modest intensity of above 1.134*10⁻²⁵ cm⁻¹/(molecule·cm⁻²). There is a second measurable CO₂ line located at 1912.69 cm⁻¹ with intensity of 1.009×10⁻²⁶ cm⁻¹/(molecule·cm⁻²). Also measured simultaneously along with CO₂ and NO is H₂O that has a strong absorption line located at 1912.5 cm⁻¹ with an intensity of 1.110×10⁻²³ cm⁻¹/(molecule cm⁻²). This unambiguous absorption feature is visible in exhaled breath and in ambient air. The 5.2 μm region contains adequate separation between NO, CO₂ and H₂O lines due mainly to the narrow spectral line widths of IV-VI laser emission. The NO and CO₂ absorption lines of interest between 1870 cm⁻¹ to 1940 cm⁻¹ obtained from the HITRAN database are shown in FIG. 3. There exist other possible candidate NO and CO₂ absorption lines for simultaneous measurements without interference from each other between 1895 cm⁻¹ and 1925 cm⁻¹, which are adequate for the eNO breath analysis procedure as described herein.

[0057] The TLAS system 41 is shown in FIG. 4. A single IV-VI laser 42 (Ekips Technologies, Norman, Okla.) with typical optical output power of 300 μW was mounted to a temperature-controlled stage not shown inside sealed cryostat 46 kept at cryogenic temperatures using a closed-cycle cryogenic refrigerator, not shown, rated for continuous maintenance free operation (CryoTiger™, APD Cryogenics, Allentown, Pa.) and pumped by ion pump 43. A laser beam represented by line 48 emitted from the IV-VI laser was first directed through a ZnSe window, not shown, and onto off-axis-parabolic mirror (OAPM) 50 to collimate the beam. A combination of flat and concave mirrors 52 was used to direct beam 48 through a 107-meter multipass White cell 54 (Infrared Analysis, Anaheim, Calif.). Upon exiting White cell 54, beam 53 was focused using ZnSe lens 55 and passed through a ZnSe window not shown onto HgCdTe mid-IR photovoltaic detector 56 also located inside cryostat 46. An integrated heater and temperature controller (Lakeshore, Westerville, Ohio), not shown, maintained stable laser operating temperatures at 102 K with an accuracy of ±0.01 K.

[0058] A low noise laser current driver in current driver and function generator assembly 58 controlled by personal computer 64 as indicated schematically by arrow 61 supplied currents between 800 mA and 900 mA. A sawtooth voltage ramp of 40 Hz and 0.11 V_(pp) was used to tune the single mode laser emission from 1912.5 cm⁻¹ to 1913.0 cm⁻¹. Superimposed onto the sawtooth ramp is a smaller triangle waveform at 26.5 kHz and 0.01 V^(pp) to modulate the laser emission frequency. The output of photovoltaic detector 56 is pre-amplified before a commercial lock-in amplifier 60 (Stanford Research Systems, Sunnyvale, Calif.) sampled the signal at twice the modulation frequency, a scheme known as second harmonic (2f) detection. A TTL signal from the 40 Hz ramp waveform generator was used to trigger the analog-to-digital (A/D) acquisitions of the output signal from the lock-in amplifier. Personal computer 64 controlled a 12-bit A/D converter card (National Instruments, Austin, Tex.) and acquired 500 data points per scan at a sampling frequency of 20 kHz. To reduce high frequency noise, 75 consecutive scans were co-averaged and then sent through a digital low pass Butterworth filter. The largest source of optical noise in the system was etalon fringes originating in White cell 54 and system optics.

[0059] Spectral shifting of the spectrum can occur due to slight temperature variations of the heat sink for the laser. To counteract this shifting effect, the H₂O absorption peak at 1912.5 cm⁻¹ shown in FIG. 8, was used as a spectral reference to line up each spectrum before co-averaging to reduce smear and improve detection sensitivities. A custom software program was used to control the external functionalities of lock-in amplifier 60, function generators and current driver part of assembly 58 using IEEE-488.2 GPTB communications. The software also performed the co-averaging, filtering, and spectral analysis algorithms for determining concentrations based on breath eNO/eCO₂ ratios. A second harmonic spectrum of human breath containing peaks for NO, CO₂, H₂O, and associated HITRAN absorption line strengths is shown in FIG. 8.

[0060] Second harmonic spectra contain absorption magnitudes that are directly proportional to concentrations of the associated molecular species. A calibration curve of the instrument was generated using a gas dilution system, which is designed for diluting a 10 ppm±2% NO gas standard (Airgas, Mobile, Ala.) with purified N₂. Mass flow controllers located at the inlet to the White cell were used to mix various flows of NO with N₂ to achieve continuous flow concentrations from 10 ppm down to 20 ppb. To quantify concentrations, a comparison spectrum was collected at a known concentration of 50 ppb. The absorption magnitude of a 50 ppb NO comparison spectrum was then compared to subsequent absorptions of NO at diluted concentrations down to 20 ppb using a least squares fitting routine which will subsequently be described in detail. The least-squares fitting routine returned the average absorption magnitude over the entire spectral feature including both negative lobes, which is more accurate than just measuring the peak of the absorption feature. The calibration curve showing diluted NO gas standard calibrations vs. measured NO is shown in FIG. 9. The error bars for the points in FIG. 9 represent the standard deviation over 200 consecutive data points measured at each calibration concentration. The line shapes at differing concentrations are shown in FIG. 10. Measured NO absorption magnitudes have a strong linear relationship (R²=0.998) with calibrated NO concentrations. The minimum detection limit for a 4 second integration time (75 co-adds) was determined to be 1.5 ppb based upon the V_(RMS) noise in the baseline of the second harmonic spectrum. Further improvement of this figure of merit is possible using faster electronics to collect more spectra in a given time period.

[0061] Breath measurements were performed at a pressure of 13 Torr to reduce line broadening and interference between NO, CO₂ and H₂O. Mechanical vacuum pump 64 including pump exhaust 65 shown in FIG. 10 induced flow through the gas cell at a constant rate of 2 L/minute using flow controllers, not shown. This rate of gas suction was comfortable for patients exhaling into the system over a period of 20 seconds or less. The breath collection device (Quintron, Milwaukee, Wis.) was designed to collect single-exhalations and consisted of T piece 66 connected to disposable mouthpiece 68, 500 mL discard bag 70, and ¼″ diameter Teflon tubing 72 directing breath through flow controllers not shown and into White cell 54. The discard bag accepted the first 500 mL of breath at little to no breathing resistance. This headspace breath contains a high concentration of NO originating from the nasal cavity. The remaining exhaled air enters the Teflon tubing at a constant rate of 2 L/min. Volunteers were instructed to exhale a single breath with force, which assisted in closing the velopharyngeal aperture limiting the entry of nasal NO via the posterior nasopharynx. A one-way flutter valve not shown located at the entrance to the discard bag prevented headspace breath from re-entering the breath collection system, and the discard bag was manually emptied after each exhalation. All breath measurements given in this report are single breath exhalations for 20 seconds unless otherwise stated. Institutional Review Board approval was granted from the University of Oklahoma for human subjects research and each participant signed an informed consent form prior to donating breath.

[0062] The magnitude of absorption due to breath eNO and eCO₂ was determined using a least-squares fitting routine, which uses a comparison spectrum to analyze measured spectra during breath testing. Two comparison spectra, denoted as #1 and #2, at unknown different concentrations of NO and CO₂ are shown in FIG. 11. The rectangular windows encompassing the NO and CO₂ absorption features define the windows 150 and 152, respectively, used to compare the comparison spectra to the measured spectra and encompass both negative lobes and the absorption peak. To characterize the relationship between the NO and CO₂ absorption profiles within each comparison, background spectra were subtracted from each comparison spectrum to eliminate any baseline offset. Next, the peak absorption voltages were determined to obtain a voltage ratio, (V_(NO)/V_(CO) ₂ ).

[0063] During breath donations, measured spectra containing the absorption features for eNO and eCO₂ were compared to either comparison spectrum #1 or #2 using the least squares fitting routine. The measured absorption line features contained in the set window for the sample (Y) and the comparison (X) have a linear relationship of the form Y_(j)=a+bX_(j). Here, X and Y are the measured voltage amplitudes within a window containing the entire absorption feature including the negative lobes and some baseline on either side. The amplitude scaling factor, b, represents the absorption magnitude determined using the least squares method shown in Equation (1). The coefficient, a, represents baseline offset and is ignored. The index, j, in Equation (1) represents the position of the channel number in acquired spectra as shown in FIG. 10. $\begin{matrix} {b = \frac{{\left( {\sum Y_{j}} \right)\left( {\sum X_{j}} \right)} - {N\left( {\sum{X_{j}Y_{j}}} \right)}}{\left( {\sum X_{j}} \right)^{2} - {N\left( {\sum X_{j}^{2}} \right)}}} & (1) \end{matrix}$

[0064] Example eNO and eCO₂ absorption magnitude arrays over time obtained for 20-second breath donations from a nonasthmatic and asthmatic volunteer are shown in FIG. 12. The end-tidal (or maximum) values in the plot of FIG. 12 occur after the end of the exhalation period because of short delays from gas exchange and software processing overhead. Prior to exhalation, 5 seconds of NO and CO₂ measurements are performed and averaged to determine their absorption magnitudes in the ambient air. The absorption magnitude for eCO₂, b_(eCO) ₂ , is determined by taking the end-tidal value in the exhalation trend array, [b_(eCO) ₂ ]_(i). The index, i, in the exhalation trend array denotes absorption magnitude data points collected over the breath analysis period. The maximum b_(eCO) ₂ value is used to verify correct breath donation. This works as a good verification because eCO₂ concentrations are always greater than CO₂ concentrations in the ambient air. The absorption value in the [b_(eNO)]_(i) array measured during the exhalation period that most deviates from the established baseline absorption magnitude for NO in the ambient is used to determine b_(eNO). For determining b_(eNO), it is not proper to use only the maximum value in [b_(eNO)]_(i) because it is possible to have larger NO concentrations in the ambient air than in exhaled breath. Once b_(eNO) and b_(eCO) ₂ have been determined, equation (2) is used to describe the overall absorption ratio, A_(eNO)/A_(eCO) ₂ , relating the measured absorption magnitudes of analyzed breath samples to the voltage magnitudes of the comparison spectra. $\begin{matrix} {\frac{A_{eNO}}{A_{{eCO}_{2}}} = {\frac{b_{eNO}}{b_{{eCO}_{2}}} \times \frac{V_{NO}}{V_{{CO}_{2}}}}} & (2) \end{matrix}$

[0065] Utilizing known standard absorption line strengths (S(ν)) and pressure broadening coefficients (g) found in the HITRAN database, equation (3) can be used to relate the concentrations of eNO and eCO₂ in breath, where C_(eNO) and C_(eCO) ₂ represent the concentration of eNO and eCO₂, respectively. $\begin{matrix} {C_{eNO} = {\left( \frac{A_{eNO}}{A_{{eCO}_{2}}} \right)\left( \frac{g_{NO}}{g_{{CO}_{2}}} \right)\left( \frac{{S(\upsilon)}_{{CO}_{2}}}{{S(\upsilon)}_{NO}} \right) \times C_{{eCO}_{2}}}} & (3) \end{matrix}$

[0066] Equation (3) was derived using Beer's law and the fact that second harmonic spectra produce absorption magnitudes that have an approximate linear relationship with the concentration of the absorbing gas species. Equation (3) assumes that laser power is equivalent across both the NO and CO₂ absorption lines. To solve for C_(eNO), it is assumed that eCO₂ concentrations are 4% since the typical value for exhaled C_(eCO) ₂ in human breath is in the range of from about 4% to about 5%. Equation (3) is vulnerable to error if actual C_(eCO) ₂ from an individual significantly deviates from the foregoing range. Slight variations (±10%) in actual C_(eCO) ₂ , however, do not significantly affect the interpreted results because there is not a critical clinical importance at this time in obtaining high precision eNO concentrations. A 10% variation in eCO₂ concentration would give a typical error of about ±2 ppb in the calculated eNO concentration, which is much smaller than the difference between the eNO concentration ranges for asthmatics (30 ppb to 80 ppb) and nonasthmatics (5 to 20 ppb).

[0067] Results of testing five individuals (four nonasthmatics and one asthmatic) using comparison spectra #1 and #2 over a period of 10-days are presented. Calibrated eNO levels are compared to calculated eNO concentrations using equation (3). Also given are the results of studying different breath testing parameters including differing exhalation times, White cell pressures, and ambient NO levels.

[0068] For comparison spectrum #1, each participant gave three breaths and the calculated eNO results using equation (3) were averaged over the three breaths. The same procedure was repeated using comparison spectrum #2. To perform calibration measurements for each participant, a diluted NO standard of 100 ppb was flowed through White cell 54 for 20 seconds, just as if a participant was exhaling. FIG. 13 shows a representative example concentration trend for calibrated NO flowing through the gas cell. The 100 ppb NO signal over the 20 second period was compared to a reference spectrum collected while White cell 54 was saturated with 100 ppb NO and the associated average absorption magnitude (b) was ˜1.0, as indicated in FIG. 13. The 100 ppb NO sample flow for 20 seconds does not completely saturate the White cell volume of 16 L at a gas exchange rate of 2 L/min, and a small software time constant due to data computational overhead does not allow b_(cal eNO) to completely reach 1.0. Immediately following analysis of the 100 ppb NO calibration gas, the volunteer exhaled into the system and the absorption magnitude for b_(eNO) was then compared to the 20 second 100 ppb calibration NO absorption magnitude, b_(cal NO). The calibrated eNO concentration, C_(cal eNO), was calculated using equation (4). $\begin{matrix} {C_{caleNO} = {\left( \frac{b_{eNO}}{b_{calNO}} \right) \times \left( {100{ppb}\quad {Reference}\quad {NO}} \right)}} & (4) \end{matrix}$

[0069] Three sequential calibrated eNO breath measurements were performed and averaged to determine the calibrated eNO concentration, C_(cal NO). It should be noted that a 16 L cell volume is suitable when discarding headspace, but smaller cell volumes are more desirable for rapid gas exchange rates and improved temporal resolution.

[0070] The five participants donated nine breaths daily over the period of ten working days (three breaths for comparison spectrum #1, three breaths for comparison spectrum #2, and three breaths for calibration). The relationship between calibrated eNO results vs. the calculated eNO results for comparison spectra #1 and #2 over the ten-day period are shown in FIG. 14 with error bars. There was a good linear relationship (R²=0.939) between the two methods showing that equation (3) using a 4% value for eCO₂ concentrations allowed accurate eNO measurements over the two-week testing period.

[0071] Adults found the 20 second exhalation time comfortable using a constant 2 L/min flow rate. The young, elderly, or ill, however, may find a 20 second exhalation period too long. FIG. 15 shows the results of an adult participant exhaling for periods of 15, 10 and 5 seconds, which would simulate breath collection from a child or adult with limited lung function. The longer the exhalation times, the stronger the measured signals due to more NO and CO₂ molecules occupying White cell 54. However, the eNO and eCO₂ ratios together help to compensate for variations in exhalation times and resulted in little variation in calculated eNO concentrations using equation (3), as shown in Table I.

[0072]FIG. 16 shows eCO₂ trends from an individual exhaling at sample cell pressures ranging from 13 Torr to 33 Torr. As the pressure increases, the volume increases and the trends have a less apparent plateau because breath is not filling as much of the White cell volume. FIG. 17 shows the calculated eNO results versus pressure. Varying exhalation times and cell pressures do not appear to affect calculated eNO results significantly.

[0073] During the early morning and evening rush hours, NO levels in the ambient air have been observed to be as high as 200 ppb, due mainly to automobile exhaust. To test the effect of elevated ambient NO levels on measured eNO values, a volunteer donated breath in the morning when ambient NO levels were high, above 20 ppb, and again in the mid-afternoon when ambient levels were below 5 ppb. FIGS. 18(a) and 18(b) show the eNO and eCO₂ trends for both breath donations from the same volunteer. When NO concentrations in ambient air are larger than eNO in breath, breath eNO displaces ambient NO in the cell and the total concentration of NO in the cell goes down. Lower eNO concentrations than ambient NO concentrations suggest inhaled NO from the ambient air is rapidly absorbed by airway tissues and not subsequently exhaled. Recent in vivo measurements of NO and its chemical reaction products in human airways show that NO rapidly consumes reactive oxidative species (ROS) producing less reactive intermediate compounds such as ONOO— and ONOOH. This process leads to the accumulation of the innocuous product NO₃—. It is suggested that high eNO levels in the breath of asthma sufferers may actually be associated with a protective mechanism in which production of endogenous NO reduces the concentration of more damaging ROS that are also produced in the airways of individuals with asthma. The observation of exhaled NO concentrations that are lower than ambient levels shows that ambient (exogenous) NO is also rapidly consumed by airway tissues. It is not clear whether or not such consumption in the lungs of healthy or asthmatic individuals is beneficial.

[0074] Elevated ambient NO acts as a source of interference when using chemiluminescence; however, the breath collection method of the invention combined with a mid-IR TLAS system provided for repeatable eNO measurements regardless of high or low ambient NO concentrations. Since ambient NO concentrations are not a factor influencing reproducibility, the instrument may be operated in environments where air pollution effects are significant.

EXAMPLE 2

[0075] The following example describes how the breath analysis method and apparatus of the invention may be used in a clinical setting to diagnose and monitor the efficacy of treatment of an asthmatic patient with anti-inflammatory therapy.

[0076] In FIG. 19, lower plot 162 shows eNO concentrations for a healthy volunteer indicating that eNO is below 20 ppb as expected in the absence of disease. Upper plot 160 shows eNO concentrations for a 42-year-old white male who initially did not carry a diagnosis of asthma. He did have a history of severe seasonal allergies including allergic rhinitis. The patient did experience intermittent chest “heaviness”, though he denied more obvious symptoms of the disease. His initial spirometry was normal, however, subsequent methacholine challenge testing was positive, indicating hyperactive airways and a likely diagnosis of asthma. The patient's eNO was found to be elevated (>40 ppb) and subsequently a trial of inhaled glucocorticoids was undertaken. As illustrated in FIG. 19, by the nine-day mark, the patient's eNO had dramatically fallen and was in the normal range of below 20 ppb. Despite his initial lack of symptoms, the patient subjectively felt much better when treated with the inhaled glucocorticoids.

[0077] Methacholine is a medication that will induce airway obstruction only in the presence of “hyper-reactive” airways. A positive test is indicated by a 20% fall in the measured baseline FEV1 (forced expired volume in one second). This has been the “gold standard” for establishing the diagnosis of asthma, a disease that can be quite variable in its presentation. Despite its utility, the methacholine challenge test is time consuming (˜1 hour), cumbersome, expensive to perform, and potentially risky because a bronchoconstrictive drug is administered. It certainly is not suitable for routine monitoring of the asthmatic. Unlike the methacholine test, the eNO test is fast, easy to perform, economical, and presents essentially no risk to the patient. It provides an assessment of underlying airway inflammation, which is a chronic condition in asthma patients. Other asthma diagnostic tests such as spirometry and peak flow measurement evaluate the airway constrictive component of the disease, a typically acute condition which may not be present during a clinical evaluation. The breath analysis method and apparatus of the invention and its ability to determine real-time eNO concentrations with ppb sensitivities provides a reliable clinical tool for the diagnosis and monitoring of asthma. 

What is claimed is:
 1. A method for measuring the concentration of a marker gas in a gas mixture comprising steps of; obtaining a gas mixture sample initially including said marker gas and a calibration gas having an independently known concentration and inherently present in said gas mixture for a gas mixture sampling interval; providing a spectrometer including a spectrometer light source, a spectrometer gas sample cell, a spectrometer detector and a spectrometer computer; placing said gas mixture sample in said spectrometer gas sample cell; illuminating said gas mixture sample with said spectrometer light source; detecting a transmitted light beam which has passed through said gas mixture sample with said spectrometer detector to produce a detector electronic signal sequence; transmitting said detector electronic signal sequence to said spectrometer computer wherein said computer includes an executable program; acquiring said detector electronic signal sequence using said spectrometer computer for a signal acquisition interval to obtain a gas mixture spectrum; storing said gas mixture spectrum; analyzing said gas mixture spectrum using said spectrometer computer said step of analyzing further including steps of; identifying a marker gas absorption feature and a calibration gas absorption feature, determining a marker gas absorption feature intensity and a calibration gas absorption feature intensity, storing said marker gas absorption feature intensity and storing said calibration gas absorption feature intensity as a function of time over said gas mixture sampling interval; repeating said steps of acquiring and analyzing for multiple signal acquisition intervals to identify a single signal acquisition interval wherein a known concentration calibration gas absorption feature which corresponds to an independently known calibration gas concentration and a simultaneous marker gas absorption feature occur; calculating a ratio of said simultaneous marker gas absorption feature intensity to said known concentration calibration gas absorption feature intensity and multiplying said ratio by a proportionality constant to determine concentration of said marker gas.
 2. The method of claim 1 wherein said gas mixture sample is exhaled breath.
 3. The method of claim 1 wherein said obtaining step further includes keeping said spectrometer gas sample cell at a pressure selected to reduce line broadening and interference between said marker gas absorption feature and said calibration gas absorption feature.
 4. The method of claim 2 wherein said exhaled breath simultaneously includes a biomarker gas characteristic of a disease and a calibration gas.
 5. The method of claim 4 wherein said biomarker gas is selected from the group consisting of NO, H₂O₂, CO, CO₂, CS₂, C₂H₆, CH₃O, NH₃, H₂S, and OCS.
 6. The method of claim 4 wherein said calibration gas is selected from the group consisting of CO₂, H₂O, N₂O and CH₄.
 7. The method of claim 1 wherein said spectrometer is a mid-infrared tunable laser absorption spectroscopy system.
 8. The method of claim 7 wherein said laser is a IV-VI diode laser further characterized by an emission wavelength in the range of from about 3 μm to about 10 μm.
 9. The method of claim 1 further including placing said gas mixture sample in a Herriott cell.
 10. The method of claim 1 further including placing said gas mixture sample in a multi-pass White cell.
 11. The method of claim 1 wherein said gas mixture spectrum is a second harmonic absorption spectrum.
 12. The method of claim 1 wherein said acquiring step further includes steps of obtaining at least two detector electronic signal sequences, storing said detector electronic signal sequences to obtain stored detector electronic signal sequences, performing a running co-average of said stored detector electronic signal sequences to obtain co-averaged detector signal sequences, and digitally filtering said co-averaged detector signal sequences to generate a gas mixture spectrum.
 13. A method for assessing human airway inflammation comprising steps of; collecting an orally exhaled human breath sample wherein said orally exhaled human breath sample further includes exhaled NO and exhaled CO₂ for an exhalation interval; providing a spectrometer including a spectrometer light source, a spectrometer gas sample cell, a spectrometer detector and a spectrometer computer; placing said orally exhaled human breath sample in said spectrometer gas sample cell; illuminating said orally exhaled human breath sample with a spectrometer light source; detecting a transmitted light beam which has passed through said orally exhaled human breath sample with said spectrometer detector to produce a detector electronic signal sequence; transmitting said detector electronic signal sequence to said spectrometer computer wherein said computer includes an executable program; acquiring said detector electronic signal sequence using said spectrometer computer for a signal acquisition interval to obtain a human breath sample spectrum; analyzing said human breath sample spectrum using said spectrometer computer, said step of analyzing further including steps of; identifying an exhaled NO absorption feature and an exhaled CO₂ absorption feature, determining an exhaled NO absorption feature intensity and an exhaled CO₂ absorption feature intensity, storing said exhaled NO absorption feature intensity and said exhaled CO₂ absorption feature intensity as a function of time; repeating said acquiring and analyzing steps for multiple signal acquisition intervals to identify a single signal acquisition interval wherein a known concentration CO₂ gas absorption feature which corresponds to an independently known CO₂ gas concentration and a simultaneous NO absorption feature occur; calculating a ratio of said simultaneous NO absorption feature intensity to said known concentration CO₂ gas absorption feature intensity and multiplying said ratio by a proportionality constant to obtain concentration of said exhaled NO thereby providing an assessment of human airway inflammation.
 14. The method of claim 13 wherein said collecting step further includes keeping said spectrometer gas sample cell at a pressure selected to reduce line broadening and interference between said exhaled NO absorption feature and said CO₂ absorption feature in said orally exhaled human breath sample.
 15. The method of claim 13 wherein said step of collecting further includes having a human subject orally exhale a single breath as said human subject would normally exhale including initial exhaled breath and remaining exhaled breath into said spectrometer gas collection chamber wherein said spectrometer gas collection chamber further includes a spectrometer gas cell for said remaining exhaled breath and a discard container to accept said initial exhaled breath thereby preventing said initial exhaled breath from entering said spectrometer gas sample cell.
 16. The method of claim 13 wherein said spectrometer is a mid-infrared tunable laser absorption spectroscopy system.
 17. The method of claim 16 wherein said laser is a IV-VI diode laser further characterized by an emission wavelength in the range of from about 3 μm to about 10 μm.
 18. The method of claim 13 further including placing said orally exhaled human breath mixture sample in a Herriott cell.
 19. The method of claim 13 further including placing said orally exhaled human breath sample in a multi-pass White cell.
 20. The method of claim 13 wherein said step of placing said orally exhaled human breath in said spectrometer further includes introducing said orally exhaled human breath sample into said spectrometer gas sample cell and pumping to induce a flow through said spectrometer gas sample cell.
 21. The method of claim 13 wherein said human breath sample spectrum is a second harmonic absorption spectrum.
 22. The method of claim 13 wherein said step of acquiring further includes steps of obtaining at least two detector electronic signal sequences, storing said detector electronic signal sequences to obtain stored detector electronic signal sequences, performing a running co-average of said stored detector electronic signal sequences to obtain co-averaged detector signal sequences, and digitally filtering said co-averaged detector signal sequences to generate said human breath sample spectrum.
 23. The method of claim 13 wherein said orally exhaled human breath is allowed to flow through said spectrometer gas sample cell at a first flow rate and at a second flow rate.
 24. The method of claim 23 wherein said first flow rate is greater than said second flow rate and said first flow rate is used before said second flow rate.
 25. The method of claim 13 further including identifying a CO₂ absorption feature intensity maximum value and a simultaneous NO absorption feature intensity value obtained simultaneously during a single signal acquisition interval and using said CO₂ absorption feature intensity maximum value and said simultaneous NO absorption feature intensity value in said calculating a ratio step.
 26. The method of claim 25 wherein said CO₂ absorption feature intensity maximum value is associated with an independently known CO₂ concentration and said independently known CO₂ concentration is in the range of from about 4% to about 5%.
 27. The method of claim 13 wherein said concentration of exhaled NO is in the range of from about 5 parts per billion to about 100 parts per billion and indicates airway inflammation.
 28. An apparatus for measuring the concentration of a marker gas in a gas mixture sample initially including said marker gas and a calibration gas having an independently known concentration and inherently present in said gas mixture comprising; a spectrometer light source for illuminating said gas mixture sample, a spectrometer gas sample cell including said gas mixture sample, a spectrometer detector for detecting light transmitted through said gas mixture sample so that a detector signal sequence is generated, and a spectrometer computer wherein said spectrometer computer further includes an executable program for repeatedly acquiring and analyzing said detector electronic signal sequence for multiple signal acquisition intervals to obtain gas mixture spectra, storing said gas mixture spectra, analyzing said gas mixture spectra by identifying a marker gas absorption feature and a calibration gas absorption feature, determining a marker gas absorption feature intensity and a calibration gas feature intensity, calculating marker gas absorption feature intensity and calibration gas absorption feature intensity as a function of time to identify a single signal acquisition interval wherein a known concentration calibration gas absorption feature which corresponds to an independently known calibration gas concentration and a simultaneous marker gas absorption feature occur; calculating a ratio of said simultaneous marker gas absorption feature intensity to said known concentration calibration gas absorption feature intensity and multiplying said ratio by a proportionality constant to determine concentration of said marker gas.
 29. The apparatus of claim 28 wherein said gas mixture sample is exhaled breath.
 30. The apparatus of claim 28 wherein said spectrometer gas sample cell is kept at a pressure selected to reduce line broadening and interference between said marker gas absorption feature and said calibration gas absorption feature.
 31. The apparatus of claim 29 wherein said exhaled breath simultaneously includes a biomarker gas characteristic of a disease and a calibration gas.
 32. The apparatus of claim 31 wherein said biomarker gas is selected from the group consisting of NO, H₂O₂, CO, CO₂, CS₂, C₂H₆, CH₃O, NH₃, H₂S, and OCS.
 33. The apparatus of claim 31 wherein said calibration gas is selected from the group consisting of CO₂, H₂O, N₂O and CH₄.
 34. The apparatus of claim 28 wherein said spectrometer is a mid-infrared tunable laser absorption spectroscopy system.
 35. The apparatus of claim 34 wherein said laser is a IV-VI diode laser further characterized by an emission wavelength in the range of from about 3 μm to about 10 μm.
 36. The apparatus of claim 28 wherein said spectrometer gas sample cell is a Herriott cell.
 37. The apparatus of claim 28 wherein said spectrometer gas sample cell is a multi-pass White cell.
 38. An apparatus for assessing human airway inflammation comprising; a spectrometer gas sample cell including an orally exhaled human breath sample containing exhaled NO and exhaled CO₂, a spectrometer light source for illuminating said orally exhaled human breath sample, a spectrometer detector for detecting light transmitted through said orally exhaled human breath sample so that a detector signal sequence is generated, and a spectrometer computer wherein said spectrometer computer further includes an executable program for repeatedly acquiring and analyzing said detector electronic signal sequence for multiple signal acquisition intervals to obtain human breath sample spectra, storing said human breath sample spectra, analyzing said human breath sample spectra by identifying an exhaled NO absorption feature and an exhaled CO₂ absorption feature, determining an exhaled NO absorption feature intensity and an exhaled CO₂ absorption feature intensity, storing said exhaled NO absorption feature intensity and said exhaled CO₂ absorption feature intensity as a function of time, repeating said steps of acquiring and analyzing for multiple signal acquisition intervals to identify a single signal acquisition interval wherein a known concentration CO₂ gas absorption feature which corresponds to an independently known CO₂ gas concentration and a simultaneous NO absorption feature occur; calculating a ratio of said simultaneous NO absorption feature intensity to said known concentration CO₂ gas absorption feature intensity and multiplying said ratio by a proportionality constant to obtain concentration of said exhaled NO thereby providing an assessment of human airway inflammation.
 39. The apparatus of claim 38 further including a mouthpiece for accepting said orally exhaled human breath sample, a T-piece connected to said mouthpiece, and wherein said T-piece is further connected to a discard container to accept initial orally exhaled human breath and to a tube for transporting remaining orally exhaled human breath into said spectrometer gas sample cell.
 40. The apparatus of claim 39 further including a one-way flutter valve separating said T-piece from said discard container to keep initial orally exhaled human breath in said discard container.
 41. The apparatus of claim 39 further including a vacuum pump connected to said spectrometer gas sample cell for inducing flow of said remaining orally exhaled human breath through said tube and spectrometer gas sample cell.
 42. The apparatus of claim 38 wherein said spectrometer is a mid-infrared tunable laser absorption spectroscopy system.
 43. The apparatus of claim 42 wherein said laser is a IV-VI diode laser further characterized by an emission wavelength in the range of from about 3 μm to about 10 μm.
 44. The apparatus of claim 38 wherein said spectrometer gas sample cell is a Herriott cell.
 45. The apparatus of claim 38 wherein said spectrometer gas sample cell is a multi-pass White cell.
 46. The apparatus of claim 38 wherein said executable program further includes instructions for identifying a CO₂ absorption feature intensity maximum value and a simultaneous NO absorption feature intensity value obtained simultaneously during a single signal acquisition interval and using said CO₂ absorption feature intensity maximum value and said simultaneous NO absorption feature intensity value to calculate said ratio.
 47. The method of claim 46 wherein said CO₂ absorption feature intensity maximum value is associated with an independently known CO₂ concentration and said independently known CO₂ concentration is in the range of from about 4% to about 5%.
 48. A method for measuring NO concentration in orally exhaled human breath comprising steps of; having a human subject normally orally exhale a single breath, including initial orally exhaled human breath and remaining orally exhaled human breath into a mouthpiece; collecting said initially exhaled human breath in a discard container; discarding said initially orally exhaled human breath; flowing said remaining orally exhaled human breath from said mouthpiece to an NO analysis system through a tube connected to a vacuum pump and to said NO analysis system; analyzing said remaining orally exhaled human breath to obtain said NO concentration.
 49. The method of claim 48 wherein in said step of flowing said orally exhaled human breath, said vacuum pump is operated to produce a vacuum in the range of from about 10 Torr to about 80 Torr.
 50. The method of claim 48 wherein in said step of flowing said orally exhaled human breath, a flow rate in the range of from about 0.5 liters per minute to about 30 liters per minute is induced.
 51. The method of claim 48 wherein said NO analysis system is a light absorption spectrometer.
 52. The method of claim 51 wherein said light absorption spectrometer system is a mid-infrared tunable laser absorption spectrometer.
 53. An apparatus for measuring NO concentration in orally exhaled human breath comprising; a mouthpiece for accepting orally exhaled human breath, including initially exhaled human breath and remaining exhaled human breath from a human subject; a discard container connected to said mouthpiece for collecting said initially exhaled human breath; a flow tube connected to said discard container and said mouthpiece and to a vacuum pump for delivering said remaining orally exhaled human breath to an NO analysis system for obtaining said NO concentration.
 54. The apparatus of claim 53 further including a one-way flutter valve to keep initially orally exhaled human breath in said discard container.
 55. The apparatus of claim 53 wherein said NO analysis system is a light absorption spectrometer.
 56. The apparatus of claim 55 wherein said light absorption spectrometer system is a mid-infrared tunable laser absorption spectrometer.
 57. A method for measuring NO concentration in orally exhaled human breath comprising steps of; having a human subject normally orally exhale a single breath into a mouthpiece; flowing said single breath to a light absorption spectrometer system through a tube connected to a vacuum pump and to said light absorption spectrometer system; analyzing said single breath with said light absorption spectrometer system to obtain said NO concentration.
 58. The method of claim 57 wherein in said step of flowing said orally exhaled human breath, said vacuum pump is operated to produce a vacuum in the range of from about 10 Torr to about 80 Torr.
 59. The method of claim 57 wherein in said step of flowing said orally exhaled human breath, a flow rate in the range of from about 0.5 liters per minute to about 30 liters per minute is induced.
 60. The method of claim 57 wherein said light absorption spectrometer system is a mid-infrared tunable laser absorption spectrometer.
 61. An apparatus for measuring NO concentration in orally exhaled human breath comprising; a mouthpiece for accepting orally exhaled human breath from a human subject; a flow tube connected to said mouthpiece, to a vacuum pump and to a light absorption spectrometer system for delivering said orally exhaled human breath to said NO analysis system for obtaining said NO concentration.
 62. The apparatus of claim 61 wherein said light absorption spectrometer system is a mid-infrared tunable laser absorption spectrometer.
 63. A method for measuring NO concentration in orally exhaled human breath comprising steps of; having a human subject normally orally exhale a single breath into a mouthpiece; flowing said single breath from said mouthpiece to an NO analysis system through a tube connected to a vacuum pump and to said NO analysis system first at a first flow rate and later at a second flow rate, wherein said first flow rate is greater than said second flow rate; analyzing said single breath with said NO analysis system to obtain said NO concentration.
 64. The method of claim 63 wherein in said step of flowing said orally exhaled human breath, said vacuum pump is operated to produce a vacuum in the range of from about 10 Torr to about 80 Torr.
 65. The method of claim 63 wherein in said step of flowing said orally exhaled human breath, said first flow rate is in the range of from about 10 liters per minute to about 30 liters per minute and said second flow rate is the range of from about 0.5 liters per minute to about 20 liters per minute.
 66. The method of claim 65 wherein said NO analysis system is a light absorption spectrometer.
 67. The method of claim 66 wherein said light absorption spectrometer system is a mid-infrared tunable laser absorption spectrometer.
 68. An apparatus for measuring NO concentration in orally exhaled human breath comprising; a mouthpiece for accepting a single orally exhaled human breath from a human subject; a flow tube connected to said mouthpiece, to a vacuum pump and to an NO analysis system and including a flow rate controller for sequentially generating a first flow rate and a second flow rate wherein said second flow rate is less than said first flow for delivering said single orally exhaled breath to said NO analysis system for obtaining said NO concentration.
 69. The apparatus of claim 68 wherein said NO analysis system is a light absorption spectrometer.
 70. The apparatus of claim 69 wherein said light absorption spectrometer system is a mid-infrared tunable laser absorption spectrometer. 